Salt-tolerant heterotrophic nitrification complex microbial inoculant and application thereof

Abstract

The invention discloses a salt-tolerant heterotrophic nitrification complex microbial inoculant and application thereof, the salt-tolerant heterotrophic nitrification complex microbial inoculant is formed by compounding seed solutions of halomonas alcaliphila LJK2, Mariobacter LJK14 and halomonas alcaliphila LJK7, and the volume ratio of the halomonas alcaliphila LJK2 seed solution to the Mariobacter LJK14 seed solution to the halomonas alcaliphila LJK7 seed solution is (2.0-2.5): (1.5-2.0): (1.0-1.5). The heterotrophic nitrification complex microbial inoculant provided by the invention has salinity tolerance of up to 20%, and still has denitrification efficiency of 98% or above when the salinity is up to 12%. Therefore, the heterotrophic nitrification complex microbial inoculant disclosed by the invention has a huge application prospect in the aspect of biological denitrification of the high-salinity mariculture tail water.

Description

Technical Field

[0001] The invention relates to the technical field of microbial wastewater treatment, and in particular to a salt-tolerant heterotrophic nitrifying composite bacterial agent and application thereof. Background Art

[0002] At present, the focus of domestic research on marine aquaculture tailwater treatment technology is to explore and develop new technologies, which can not only efficiently remove various pollutants in marine aquaculture tailwater, but also reduce treatment costs. Microbial treatment technology is considered to be one of the important means of marine aquaculture tailwater treatment. Traditional microbial denitrification technology mainly relies on the action of autotrophic nitrifying bacteria and anaerobic denitrifying bacteria, which is divided into two stages: aerobic and anaerobic. It converts nitrogen-containing organic matter into N2 through ammonification, nitrification and denitrification, thereby achieving the purpose of nitrogen removal. Since the growth rate of autotrophic nitrifying bacteria is relatively slow, a long culture time is required, which to a certain extent limits the widespread application of this technology. In addition, the high-salt and high-nitrogen concentration tailwater produced by marine aquaculture will significantly inhibit the enzyme activity of microorganisms, destroy the balance of their flora structure, and then lead to a significant decline in the denitrification performance of microorganisms during the tailwater treatment process. This phenomenon has largely restricted the widespread application of microbial treatment technology in marine aquaculture tailwater treatment. Therefore, seeking a high-salt tailwater denitrification microbial treatment technology that is both energy-saving and environmentally friendly requires us to find an effective solution. Summary of the invention

[0003] In view of the problems in the background technology, the present invention proposes a method for treating high-salinity seawater aquaculture tail water and NH4 + The invention discloses a salt-tolerant heterotrophic nitrifying composite bacterial agent with high -N removal rate, and also provides its application in reducing the ammonia nitrogen content in high-salt water bodies.

[0004] The present invention adopts the following technical solutions:

[0005] A salt-tolerant heterotrophic nitrifying composite bacterial agent is compounded from the seed liquid of alkaliphilic halomonas LJK2, Marinobacterium LJK14 and alkaliphilic halomonas LJK7, wherein the alkaliphilic halomonas LJK2 is deposited in the Guangdong Microbial Culture Collection Center on December 23, 2021, with a deposit number of GDMCC NO: 62157, the Marinobacterium LJK14 is deposited in the Guangdong Microbial Culture Collection Center on December 23, 2021, with a deposit number of GDMCC No: 62156, and the alkaliphilic halomonas LJK7 is deposited in the Guangdong Microbial Culture Collection Center on May 17, 2022, with a deposit number of GDMCC No: 62477; the volume ratio of the seed liquid of the alkaliphilic halomonas LJK2, the Marinobacterium LJK14 and the alkaliphilic halomonas LJK7 is 2.0-2.5:1.5-2.0:1.0-1.5.

[0006] Preferably, the volume ratio of the seed solutions of the alkaliphilic halomonas LJK2, the Marinobacterium LJK14 and the alkaliphilic halomonas LJK7 is 2.1-2.3:1.7-1.9:1.1-1.3.

[0007] Preferably, the volume ratio of the seed solutions of Halobacterium alkaliphilum LJK2, Bacillus marinoi LJK14 and Halobacterium alkaliphilum LJK7 is 2.2:1.8:1.2.

[0008] Preferably, the preparation method of the seed solution of each strain is as follows: the corresponding strain is inoculated into LB medium, cultured in a shaking table at 25-35°C and 100-200rpm for 20-30h, centrifuged the bacterial suspension in a refrigerated centrifuge at 3000-5000rpm for 5-15min, removed the supernatant, precipitated, washed the bacteria with sterile water, centrifuged again to remove the supernatant, repeated 2-3 times, and the OD value of the bacterial suspension was adjusted with sterile water. 600 The concentration was adjusted to 0.6-0.8 to obtain the seed solution of the strain.

[0009] As a general inventive concept, the present invention also provides a use of the above-mentioned salt-tolerant heterotrophic nitrifying composite bacteria agent in reducing the ammonia nitrogen content in a high-salt water body.

[0010] Preferably, the application comprises the following steps: adding a salt-tolerant heterotrophic nitrifying composite bacteria agent and a carbon source to a high-salt water body with a pH of 6 to 8, the volume ratio of the salt-tolerant heterotrophic nitrifying composite bacteria agent to the water body being 3% to 15%, and culturing for 45h to 55h.

[0011] Preferably, the ammonia nitrogen concentration in the high salt water body is 50 to 200 mg·L -1 .

[0012] Preferably, the carbon source is added to the high-salinity water body so that the C / N ratio is 30-100.

[0013] Preferably, the carbon source is sodium citrate.

[0014] Preferably, the salinity of the high-salt water body is 1% to 12%.

[0015] Compared with the prior art, the advantages of the present invention are:

[0016] The heterotrophic nitrification composite bacterial agent of the present invention can tolerate salinity up to 20%, and still has a denitrification efficiency of more than 98% when the salinity is as high as 12%, indicating that the heterotrophic nitrification composite bacterial agent of the present invention has great application prospects in biological denitrification of high-salinity seawater aquaculture tail water.

[0017] Halomonas alkaliphila LJK2 was deposited in Guangdong Microbiological Culture Collection Center (GDMCC), Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, with the deposit number GDMCC NO: 62157 and the deposit date December 23, 2021.

[0018] Marinobacter sp. LJK14 is deposited in Guangdong Microbiological Culture Collection Center (GDMCC), Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with the deposit number GDMCC No: 62156 and the deposit date is December 23, 2021;

[0019] Halomonas salifodinae LJK7 is deposited in Guangdong Microbiological Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology, with the deposit number GDMCC No: 62477 and the deposit date May 17, 2022. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to make the present invention more easily understood, the present invention will be described in more detail by referring to the specific embodiments shown in the accompanying drawings. These drawings only depict typical embodiments of the present invention and should not be considered as limiting the scope of protection of the present invention.

[0021] Figure 1 This is the colony morphology of the LJK2 strain.

[0022] Figure 2 This is a transmission electron scanning micrograph of the LJK2 strain.

[0023] Figure 3 To construct the phylogenetic tree of LJK2 based on 16S rDNA sequence homology.

[0024] Figure 4 This is the colony morphology of strain LJK14.

[0025] Figure 5 This is the bacterial morphology of strain LJK14.

[0026] Figure 6 This is the Gram staining result of strain LJK14.

[0027] Figure 7 Phylogenetic tree of LJK14 constructed based on 16S rDNA sequence homology.

[0028] Figure 8 This is the colony picture of strain LJK7.

[0029] Fig. 9 This is a diagram of the bacterial morphology of strain LJK7.

[0030] Fig.10 This is the phylogenetic tree of LJK7 constructed based on 16S rDNA sequence homology.

[0031] Fig.11 3D response surface plot for mixture designs.

[0032] Fig.12 This is a graph showing the effect of different carbon sources on the heterotrophic nitrification and denitrification performance of the composite bacterial agent.

[0033] Fig.13 Effect of different C / N ratios on the heterotrophic nitrification and denitrification performance of composite bacterial agents.

[0034] Fig.14 For different NH4 + -Effect of N concentration on the heterotrophic nitrification and denitrification performance of composite bacterial agent (C / N=10).

[0035] Fig.15 For different NH4 + -Effect of N concentration on the heterotrophic nitrification and denitrification performance of the composite bacterial agent (C / N=30).

[0036] Fig.16 This is a graph showing the effect of different salinities on the heterotrophic nitrification and denitrification performance of the composite bacterial agent.

[0037] Fig.17 This is a graph showing the effect of different pH on the heterotrophic nitrification and denitrification performance of the composite bacterial agent. DETAILED DESCRIPTION

[0038] The following describes the implementation modes of the present invention with reference to the accompanying drawings so that those skilled in the art can better understand the present invention and implement it. However, the enumerated embodiments are not intended to limit the present invention. In the absence of conflict, the following embodiments and the technical features in the embodiments may be combined with each other, wherein the same components are represented by the same figure marks.

[0039] Seed liquid preparation Luria-Bertani medium (1L): 5g yeast extract, 10g peptone, 33g sodium chloride. Inoculate the strain into LB medium, culture at 25-35°C, 100-200rpm shaking for 20-30h, centrifuge the bacterial suspension in a 3000-5000rpm refrigerated centrifuge for 5-15min, remove the supernatant, precipitate, wash the bacteria with sterile water, centrifuge again to remove the supernatant, repeat 2-3 times, and use sterile water to adjust the OD of the bacterial suspension. 600The concentration was adjusted to 0.6-0.8 to obtain the seed solution of the strain.

[0040] Heterotrophic nitrification medium (1L): (NH4)2SO4 0.47g, (CH2COONa)2·6H2O 5.63g, NaCl 33g, Weiss salt solution 50mL·L -1 .

[0041] Weiss salt solution (1L): K2HPO4·3H2O 6.54g, MgSO4·7H2O 2.5g, NaCl 2.5g, FeSO4·7H2O 0.05g, MnSO4·H2O 0.04g.

[0042] 1. Source of strains:

[0043] The present invention collected the tail water sediment of shrimp cultured in seawater from Pushu Village, Dianbai District, Maoming City, Guangdong Province, and three salt-tolerant HN-AD bacteria were selected through enrichment culture, separation and screening, namely, alkaliphilic Halomonas strain LJK2, Marinobacterium LJK14 and alkaliphilic Halomonas LJK7. The above three salt-tolerant HN-AD bacteria were used to construct a composite heterotrophic nitrifying agent, and the Design-Expert.V8.0.6.1 software was used to perform mixing design and optimize the ratio of the agent, and the influencing factors of the ammonia nitrogen removal performance of the above heterotrophic nitrifying agent were explored through experiments and the NH4 tolerance of the agent was determined. + -N concentration.

[0044] 2. Strain Identification

[0045] 2.1. Identification of LJK2 strain

[0046] Morphological identification

[0047] The isolated and purified strains were inoculated onto solid culture medium and the colony characteristics of the strains were observed after culturing for 36 h. Single colonies were picked and sent to the Institute of Urban Environment, Chinese Academy of Sciences (Xiamen) to observe the morphology and characteristics of the bacteria using a transmission electron microscope.

[0048] Figure 1 The results of colony observation showed that LJK2 was a light yellow, raised round small colony with smooth and complete edges, moist and sticky; it was a Gram-negative bacterium; Figure 2 The transmission electron microscopy results showed that the LJK2 bacteria were fusiform, straight or curved, with flagella and pili, but no capsule.

[0049] Physiological and biochemical identification

[0050] Physiological and biochemical identification tests were conducted according to the "Common Bacteria System Identification Manual" and the "Berger's Bacteria Identification Manual". The test items are shown in Table 1:

[0051] Table 1 Physiological and biochemical identification indicators

[0052]

[0053] The LJK2 strain is a Gram-negative bacterium, and its physiological and biochemical characteristics are shown in Table 2: The LJK2 strain can adapt to anaerobic and aerobic environments; the catalase and oxidase tests are both positive; the MR and VP tests are negative, indicating that the decomposition products of the strain are non-acidic substances and do not produce pyruvate; the indole test is negative, indicating that the strain does not have tryptophanase; the citrate test is positive; the starch hydrolysis test is negative, indicating that the strain does not have amylase; the results of the glucose, lactose sucrose and mannitol oxidation fermentation tests indicate that the strain can decompose glucose, lactose and sucrose, but not mannitol.

[0054] Table 2 Physiological and biochemical characteristics of LJK2 strain

[0055] Test Project result Test Project result Test Project result Catalase ﹢ No paraffin sealing ﹢ Indole assay ﹣ Oxidase ﹢ With paraffin wax seal ﹢ Sportiness ﹣ Glucose oxidative fermentation Produces acid but not gas MRI testing ﹣ 5% NaCl ﹢ Lactose oxidative fermentation Produces acid but not gas VP test ﹣ 7% NaCl ﹢ Sucrose oxidative fermentation Produces acid but not gas Citrate ﹢ 10% NaCl ﹢ Mannitol oxidative fermentation No acid or gas generation Starch hydrolysis ﹣

[0056] Note: “﹢” indicates positive, “﹣” indicates negative

[0057] Molecular Biology Identification

[0058] Take the bacterial solution cultured for 48 hours, dilute it and perform PCR amplification on its 16S rDNA. The PCR reaction system is 25μL: template DNA 1μL; primer 27F ((5'-AGAGTTTGATCCTGGCTCAG-3') 1μL; primer 1492R (5'-TACGACTTAACCCCAATCGC-3') 1μL; Premix Taq enzyme 12.5μL; sterile water 9.5μL. Reaction conditions: pre-denaturation and unwinding at 95℃ for 3min; denaturation process at 95℃ for 45s; annealing process at 55℃ for 45s; extension process at 72℃ for 45s; repeat the second step for 30 cycles; final extension at 72℃ for 7min. After 1% agarose gel electrophoresis, the product was photographed with a gel imaging system and sent to Jinweizhi Biotechnology Co., Ltd. for sequencing. The sequence of the sequencing results was subjected to Blast comparison analysis on the NCBI official website, and the phylogenetic tree was constructed using the MEGA7.0 neighbor-joining method to complete the strain homology analysis.

[0059] The phylogenetic tree of LJK2 was constructed based on 16S rDNA sequence homology. Figure 3As shown: The 16S rDNA of the LJK2 strain is clustered with Halomonas alkaliphilaX3, and the relationship is the closest. Combining the experimental results of the morphological characteristics and physiological and biochemical characteristics of LJK2, according to the "Common Bacterial System Identification Manual" and "Berger's Bacterial Identification Manual", the strain was identified as alkaliphilic halomonas and named Halomonas alkaliphila LJK2. On December 23, 2021, the bacteria were deposited in the Institute of Microbiology, Guangdong Academy of Sciences for patent preservation of the strain, and the strain number is GDMCC No.62157.

[0060] 2.2. Identification of LJK14 strain

[0061] Morphological identification

[0062] The strain LJK14 was inoculated into beef extract peptone medium, and the bacteria were picked after culturing for 12 hours. LJK14 was Gram-stained using the Gram kit of Changde Beekman Biotechnology Co., Ltd., and the strain morphology and Gram staining results were observed under a microscope. The strain LJK14 was inoculated into beef extract peptone medium and LB medium, and after culturing for 12 hours, it was sent to the Institute of Microbiology, Guangdong Academy of Sciences, and the morphological characteristics and motility of the strain were observed using a transmission electron microscope.

[0063] The morphological characteristics of strain LJK14 are as follows Figure 4 As shown: The results of colony observation showed that LJK14 was flesh-pink, raised, irregularly round colonies with smooth surface, irregular edges, moist and sticky bacteria that were difficult to pick up and had a special smell. Figure 5 As shown: LJK14 bacteria are short rod-shaped, have flagella, no pili, no capsule, and no motility.

[0064] Physiological and biochemical identification

[0065] Physiological and biochemical identification tests were performed on strain LJK14 with reference to the Bergey's Bacteria Identification Manual and the Common Bacteria System Identification Manual. The test items are shown in Table 3:

[0066] Table 3 Physiological and biochemical identification indicators of strain LJK14

[0067]

[0068] Gram staining results Figure 6Shown: strain LJK14 is a Gram-negative bacterium. The physiological and biochemical characteristics of LJK14 are shown in Table 4: strain LJK14 grows without paraffin sealing, but does not grow with paraffin sealing, indicating that it can adapt to aerobic environment but cannot grow in anaerobic environment; the gas production test is negative, indicating that strain LJK14 does not produce gas; the catalase and oxidase tests are both negative, indicating that strain LJK14 does not contain catalase and oxidase; the MR and VP tests are negative, indicating that the decomposition products of strain LJK14 are non-acidic substances and do not produce pyruvate; the indole test is negative, indicating that strain LJK14 does not have tryptophanase; the citrate test is negative, indicating that strain LJK14 cannot utilize citrate; the starch hydrolysis test is positive, indicating that strain LJK14 has amylase; the results of glucose, lactose sucrose and mannitol oxidation fermentation tests indicate that glucose, lactose, sucrose and mannitol cannot be decomposed; the salinity experiment shows that strain LJK14 can grow at 5% and 10% salinity, but does not grow at 15% salinity, and is a salt-tolerant bacterium.

[0069] After consulting Bergey's Manual of Bacterial Identification and Manual of Systematic Identification of Common Bacteria, it can be preliminarily inferred that strain LJK14 belongs to Kingdom Monera (Prokaryotes), Pseudomonadota (Pseudomonas), Gammaproteobacteria (γ-Proteobacteria), Pseudomonadales (Pseudomonadales), Marinobacteraceae (Marinobacteraceae), and Marinobacter (Marinobacilus).

[0070] Table 4 Results of physiological and biochemical identification experiments of strain LJK14

[0071] project result project result No paraffin sealing + MR determination - With paraffin wax seal - VP determination - Gas production experiment - Citrate - Oxidase - Starch hydrolysis + Catalase - Indole assay - Glucose oxidative fermentation No acid or gas generation Sportiness - Lactose oxidative fermentation No acid or gas generation 5% salinity + Sucrose oxidative fermentation No acid or gas generation 10% salinity + Mannitol oxidative fermentation No acid or gas generation 15% salinity -

[0072] Note: “+” indicates positive, “-” indicates negative

[0073] Molecular Biology Identification

[0074] The strain LJK14 was inoculated into LB medium and cultured for 48 hours. The bacterial liquid DNA was extracted using the Ezup column bacterial genomic DNA extraction kit and PCR amplification was performed. The PCR amplification reaction system is shown in Table 5. The PCR amplification reaction conditions are shown in Table 6. After 1% agarose gel electrophoresis, the PCR amplification product was photographed using a gel imaging system and sent to the Institute of Microbiology of Guangdong Academy of Sciences for 16S rDNA detection to determine the bacterial species. The sequencing results were analyzed by Blast alignment on the NCBI official website, and the phylogenetic tree was constructed using the MEGA7.0 neighbor-joining method to complete the homology analysis of strain LJK14.

[0075] Table 5 PCR amplification reaction system

[0076]

[0077]

[0078] Table 6 PCR amplification reaction conditions

[0079] PCR conditions parameter Pre-denaturation 95℃3min transsexual 95℃40s annealing 55℃40s extend 72℃40s cycle 30 times extend 72℃7min

[0080] The phylogenetic tree of strain LJK14 was constructed based on the 16S rDNA sequencing results. Figure 7 As shown: The 16SrDNA of strain LJK14 and Marinobacter sp.U13690101122-SW176 (JQ082151.1:16-1435) are clustered into one branch, with the closest relationship and a similarity of 99.93%. Combined with the morphological characteristics and physiological and biochemical characteristics of strain LJK14, the strain was identified as Marinobacter sp.LJK14. Strain LJK14 was deposited in the Institute of Microbiology, Guangdong Academy of Sciences for patent preservation of strains, and the strain number is GDMCC No.62156.

[0081] 2.3. Identification of LJK7 strain

[0082] Morphological identification

[0083] The purified target strain LJK7 was streaked onto LB solid culture medium and cultured at 30°C for 36 h to observe the colony morphology. The microscopic morphological characteristics of single colonies were observed using a transmission electron microscope (completed by the Institute of Urban Environment, Chinese Academy of Sciences, Xiamen). A small amount of bacteria was picked and identified by Gram staining to determine whether they were Gram-negative or Gram-positive bacteria.

[0084] The colony characteristics of strain LJK7 are as follows Figure 8 As shown: After the strain LJK7 was cultured on a solid plate for 2 days, round, raised, neatly edged and smooth pale yellow colonies were observed; the bacterial morphology results were as follows Fig. 9 As shown: strain LJK7 is short rod-shaped, straight or curved, with flagella and pili.

[0085] Physiological and biochemical identification

[0086] Refer to the Bergey's Bacteria Identification Manual and the Common Bacteria System Identification Manual for physiological and biochemical identification tests. The test items are shown in Table 7:

[0087] Table 7 Physiological and biochemical identification indicators

[0088]

[0089] The results of the physiological and biochemical characteristics test of strain LJK7 are shown in Table 8: The results of the catalase and oxidase tests of strain LJK7 are both positive; the results of the MR and VP tests are negative, indicating that the decomposition products of the strain are non-acidic substances and do not produce pyruvic acid; the indole test is negative, indicating that there is no tryptophanase; the citrate test is positive; the starch hydrolysis test is positive, indicating that there is amylase; strain LJK7 can decompose glucose, lactose, sucrose, sucrose and mannitol; the results of the salt tolerance test show that the results are positive under the conditions of 5% and 7% NaCl mass fraction, which indicates that strain LJK7 has good salt tolerance. According to the above characteristics, the Bergey Bacteria Identification Manual and the Common Bacteria System Identification Manual were consulted to judge strain LJK7 as Halomonas.

[0090] Table 8 Physiological and biochemical characteristics of strain LJK7

[0091] Test items result Test items result Oxidase + Citrate + Catalase + Starch hydrolysis + Glucose oxidative fermentation Produces acid but not gas Indole assay - Lactose oxidative fermentation Produces acid but not gas Sportiness - Sucrose oxidative fermentation Produces acid but not gas 5% NaCl + Mannitol oxidative fermentation Produces acid but not gas 7% NaCl + MRI testing - VP test -

[0092] Note: “﹢” indicates positive, and “-” indicates negative.

[0093] Molecular Biology Identification

[0094] The strain LJK7 was inoculated into LB liquid medium and cultured at 30°C for 48h. The bacterial solution was diluted and genomic DNA was extracted using a DNA extraction kit. This was used as a template, and the bacterial 16S rDNA universal primers (27F, 1492R) were used to amplify 16S rDNA by PCR according to the PCR reaction system in Table 9 and the reaction conditions in Table 10. The amplified product was sequenced by Jinweizhi Biotechnology Co., Ltd. after being verified by 1% agarose gel electrophoresis. After sequencing, the NCBI official website was entered, and the sequence was submitted to Blast for comparison analysis. Sequences with higher homology were selected, and the Neighbor-Joining method was used to complete the construction of the phylogenetic tree in MEGA 7.0 software.

[0095] Table 9 16S rDNA sequence amplification reaction system

[0096]

[0097] Table 10 PCR reaction conditions

[0098] step Temperature and time cycle Pre-denaturation 95℃3min 1 transsexual 95℃45s 30 annealing 55℃45s 30 extend 72℃45s 30 Final extension 72℃7min 1

[0099] The phylogenetic tree of LJK7 was constructed based on 16S rDNA sequence homology. Fig.10As shown in the figure, the 16S rDNA of LJK7 strain is clustered with Halomonas salifodinae strain ZSH30, and the relationship is the closest. Combining the morphological characteristics of LJK7 with physiological and biochemical tests, the strain was identified as Halomonas salifodinae and named Halomonas salifodinae LJK7.

[0100] 3. Preliminary study on the mixing of heterotrophic nitrifying bacteria and optimization of the combination ratio

[0101] Preparation of seed solution: LJK2, LJK14, and LJK7 were inoculated into LB medium and cultured in a shaking incubator at 30°C and 150 rpm for 24 h. The bacterial suspension was centrifuged in a refrigerated centrifuge at 4000 rpm for 10 min, the supernatant was removed, the precipitated bacteria were washed with sterile water, and the supernatant was removed by centrifugation. This was repeated 2-3 times. The OD value of the bacterial suspension was adjusted with sterile water. 600 Adjust to 0.6-0.8 as the seed solution for LJK2, LJK14, and LJK7.

[0102] A preliminary study on functional bacteria mixture: NH4 + -N concentration is 100 mg·L -1 , C / N is 30, pH is 7, and the preliminary design of mixed strains is shown in Table 11. The seed liquid of each strain was inoculated into 100 mL of heterotrophic nitrification medium according to the ratio in Table 11, and cultured in a shaker at 30°C and 150 rpm for 48 h. The above experimental treatments were repeated 3 times, and the NH4 in the system was measured every 6 h. + -N content.

[0103] Table 11 Preliminary design of mixed strains

[0104]

[0105] A preliminary study on NH4 in the culture system of three bacterial strains + -N content changes are as follows: Compared with the strains of the composite bacterial agent, the NH4 + -N removal rate was improved. Each strain combination had a NH4 + -N removal rate basically reached the maximum, among which the NH4 + -N removal rate reached 92.2%, LJK2 + LJK7 combination of NH4 + -N removal rate reached 85.2%, LJK2+LJK14+LJK7 combined NH4 + -N removal rate is 93.4%, LJK14 + LJK7 combination of NH4 + -N removal rate was 75.1%. The above results show that the combination of three aerobic denitrifying bacteria expressed good denitrification performance and can be symbiotically cultivated.

[0106] Mixing design determines the compound ratio of microbial agents: NH4 + -N concentration is 200 mg·L -1 , C / N is 15, pH is 7, inoculation volume is 6%, A (LJK2), B (LJK14), C (LJK7) are selected as optimization factors, the boundary values ​​of the inoculation volume of the three strains of seed liquid are set to 0-5mL, the Mixture mixture design experiment in Design-Expert 13 software is used, and the experiment is arranged according to the mixture design. The seed liquid of each strain is inoculated into a heterotrophic nitrification medium with a volume of 100mL according to the experimental design ratio, and cultured at 30℃, 150rpm shaker for 48h. The above experimental treatments are repeated 3 times, and the NH4 in the system is measured every 6h + -N content.

[0107] Mixture Design Model Analysis:

[0108] Using Design Expert 13 response surface software, a quadratic fit was performed on the data in Table 12 mixture design results to obtain NH4 + -N removal rate (R1) regression equation:

[0109] R1=2.84A+3.11B+3.07C+1.72AB+1.74AC+2.17BC+25.37ABC

[0110] The results of variance analysis of the regression equation are shown in Table 12: The experimental model was extremely significant (p < 0.01), AB (LJK2 + LJK14), BC (LJK14 + LJK7) and ABC (LJK2 + LJK14 + LJK7) had a significant effect on the composite bacterial agent NH4 + -N removal rate was significantly affected (p<0.01), while other factors were not significantly affected (p>0.05). Model correlation coefficient R 2 =0.94, indicating that the model equation can explain 94.7% of the response value (composite bacterial agent NH4 + -N removal rate) changes; Corrected correlation coefficient R 2 (Adj) =0.90, R 2 With R 2 (Adj) The difference is within 0.1, the correlation between the predicted value and the true value is high, the equation model has high credibility, and the regression equation has good fitting. This model can be used to analyze the relationship between the proportions of different functional bacteria in the composite microbial agent.

[0111]

[0112]

[0113]

[0114] Results Analysis The mixture design contours and 3D response surface show that the three-dimensional response surface diagram is spherical, such as Fig.11 As shown in the figure, it shows that the mixing ratio of strains LJK2, LJK14, and LJK7 has an effect on the composite bacterial agent NH4 + -N removal has an interactive effect; the presence of a "red vertex" in the contour map indicates that the proportion of the bacterial population of the composite microbial agent is within the experimental design range. + -N removal rate has a maximum value, A (LJK2) is the dominant factor, when the ratio of strain LJK2 is 42.0%, NH4 + -N removal rate is the largest. The above experimental results show that when the strain LJK2:LJK14:LJK7=2.2∶1.8∶1.2, the denitrification effect of YL bacterial agent is the best.

[0115] 4. Optimization of growth conditions of composite bacterial agents

[0116] The other conditions remained unchanged, and no carbon (no carbon source added), sodium bicarbonate, glucose, sucrose, sodium citrate, and sodium acetate were used as carbon sources, respectively, and inorganic carbon source sodium bicarbonate and carbon-free culture medium were used as controls; C / N was set to 0, 10, 30, 50, 80, 100, and 150, respectively; when C / N=10, the nitrogen source concentration was set to 60 mg / L, 80 mg / L, 100 mg / L, and 120 mg / L, respectively; when C / N=30, the nitrogen source concentration was set to 50 mg / L, 100 mg / L, 150 mg / L, and 200 mg / L, respectively; the salinity was set to 1%, 4%, 8%, 12%, 16%, 18%, and 20%, respectively; and the pH was set to 5, 6, 7, 8, 9, and 10, respectively.

[0117] The seed liquid of the three strains was compounded to form a composite bacterial agent and then inoculated into the heterotrophic nitrification medium. The volume ratio of the composite bacterial agent to the heterotrophic nitrification medium was 3% to 15%. After culturing for 45 to 55 hours, samples were taken to detect OD 600 , pH, NH4 + -N, NO2 - -N and NO3 - -N content.

[0118] Effect of carbon source on the treatment of tail water by composite bacterial agent

[0119] like Fig.12As shown in the figure: the effect of different carbon sources on the growth of composite bacterial agents. When no carbon is used as the carbon source, the biomass of the composite bacterial agent hardly grows and the denitrification rate is extremely low, indicating that the growth of the bacteria requires the addition of a certain carbon source; when sodium bicarbonate (inorganic carbon) is used as the carbon source, the growth of the composite bacterial agent is poor and the denitrification performance is not ideal, indicating that the bacteria can use this carbon source for autotrophy but the denitrification effect is poor; when glucose is used as the carbon source, the strain biomass is 0.343, and the biomass, NH4 + -N removal rate was lower than that of other carbon sources; when the carbon source was sucrose, the NH4 + -N removal rate reached 84.47%, but its biomass OD 600 The growth of the composite bacterial agent was poor; when sodium citrate and sodium acetate were used as carbon sources, the biomass of the composite bacterial agent reached 1.362 and 1.255 respectively. + -N removal rates were also as high as 98.06% and 99.46%, respectively. This result shows that sodium citrate and sodium acetate are both conducive to the growth of composite bacterial agents and can effectively utilize these two carbon sources for heterotrophic nitrification reactions. However, based on the analysis of the biomass, deammoniation nitrogen removal rate, accumulation of nitrite and nitrate of the strains when using these two carbon sources, sodium citrate was selected as the carbon source for the subsequent nitrification influencing factor exploration experiment. In summary, the utilization of different carbon sources by composite bacterial agents is as follows: sodium citrate>sodium acetate>sucrose>sodium bicarbonate>glucose>no carbon.

[0120] Effect of C / N on the Treatment of Tail Water by Composite Bacteria Agent

[0121] Effects of different C / N ratios on the growth of composite bacterial agents Fig.13 As shown in the figure: When C / N is 0, that is, there is not enough carbon source, the composite bacterial agent has no effect on NH4 + -N removal rate was extremely low and the strain hardly grew, which may be due to the lack of carbon source, which made the composite bacterial agent unable to carry out normal metabolic activities; when C / N was 150, although the carbon source was sufficient, NH4 + -N removal rate was low and biomass was low, which may be because the excessive C / N caused the composite bacteria to be unable to effectively utilize carbon sources during nitrification, or too much carbon source had an inhibitory effect on the strain, indicating that this C / N was not conducive to the growth of the composite bacteria; when C / N was 10-100, NH4 + -N removal rate was as high as 90%, and its biomass showed a trend of first increasing, then decreasing, and then increasing. Especially when C / N was 30 and 100, the biomass of the strain was 1.886 and 2.091 respectively, and its NH4 + -N removal rates were as high as 100%, indicating that the composite bacterial agent had excellent growth and denitrification capabilities under these two C / N conditions.

[0122] Effect of nitrogen source concentration on the treatment of tail water by composite bacterial agent

[0123] When C / N=10, different NH4 + Effect of -N concentration on heterotrophic nitrification and denitrification performance of composite bacterial agents Fig.14 As shown in the figure: With the increase of nitrogen source concentration, the biomass also increases accordingly, and the ammonia nitrogen removal rate is as high as more than 99% and there is almost no accumulation of nitrite, but the biomass is relatively low. When the nitrogen source concentration is 120 mg / L, the biomass reaches the highest 1.595, indicating that when the nitrogen source concentration is low (C / N=10), the ammonia nitrogen removal rate of the composite bacterial agent is better, but the growth of bacteria is general; when C / N=30, different NH4 + Effect of -N concentration on heterotrophic nitrification and denitrification performance of composite bacterial agents Fig.15 As shown in the figure: With the increase of nitrogen source concentration, the biomass and ammonia nitrogen removal rate of the composite bacterial agent both showed a trend of first increasing and then decreasing, and the strain growth was better than when C / N=10, with the biomass being 1.475, 1.944, 1.950, and 1.875, respectively, and the ammonia nitrogen removal rate being as high as 96% or more, especially when the nitrogen source concentration was 100 mg / L, the ammonia nitrogen removal rate of the composite bacterial agent was as high as 100%, indicating that when C / N=30 and the nitrogen source concentration was 100 mg / L, not only the growth of the bacteria, but also the denitrification effect was excellent. In summary, the composite bacterial agent has a strong tolerance to nitrogen source concentration, and when the nitrogen source concentration is in the range of 50 mg / L to 200 mg / L, the denitrification efficiency of the composite bacterial agent for marine aquaculture tail water is as high as 96% or more.

[0124] Effect of salinity on the treatment of tail water by composite bacterial agent

[0125] like Fig.16 As shown: The effect of different salinity on the treatment of tail water by composite bacterial agent. When the salinity is 1%, 4%, 8% and 12%, the bacterial OD 600 The biomass was relatively stable, with a small increase, and the biomass was 1.605, 1.964, 1.926, and 1.644 respectively, and NH4 + -N removal rate is as high as 99%; when the salinity is 16%, the bacterial OD 600 and NH4 + -N removal rate is reduced; under extreme conditions where the salinity reaches 20%, the growth of the strain almost stagnates. This may be because the excessively high salinity causes the bacteria to suffer severe dehydration stress, leading to their death and thus being unable to exert their nitrification capacity. The salinity is too low to provide suitable growth environment conditions, resulting in slow bacterial growth and low denitrification rate. High salinity environments generally inhibit bacterial nitrification activity, while the composite bacterial agent in this design has good salt tolerance and still has a high denitrification efficiency even when the salinity is 12%.

[0126] Previous studies have shown that LJK2 tolerates salinity of 8% in ammonia nitrogen water. When the salinity is 7%, the OD 600 and NH4 + -N removal rate decreased; LJK14 tolerated salinity of 7% in ammonia nitrogen water, and when the salinity was 6%, the bacterial OD 600 and NH4 + -N removal rate decreased; LJK7 tolerated salinity of 6% in ammonia nitrogen water, and when the salinity was 5%, the bacterial OD 600 and NH4 + The -N removal rate is reduced.

[0127] Effect of pH on the treatment of tail water by composite bacterial agent

[0128] pH is one of the main factors affecting the biological denitrification process. The pH value mainly affects the permeability of the cell membrane by changing the charge of the cell membrane, which in turn has a profound impact on the growth characteristics of microorganisms, the activity of metabolic enzymes, and the absorption and conversion of nutrients. The results of the influence of different pH on the effect of composite bacterial agents on tail water treatment are as follows: Fig.17 As shown in the figure: when the pH is 5, the composite bacterial agent cannot grow normally due to the low pH, so that the biomass and denitrification rate are almost 0; when the pH is 6, 7 and 8, the biomass tends to be stable, and the denitrification rate is as high as 100%, indicating that the suitable pH range for bacterial growth is 6-8; when the pH is 9, the biomass of the composite bacterial agent is lower than the previous one, but the denitrification rate is still very high; when the pH is 10, due to the high pH, ​​some enzymes inside the composite bacterial agent are inactivated, resulting in the biomass of the composite bacterial agent lower than when the pH is 9, indicating that this pH is not suitable for the growth of the composite bacterial agent. Too high or too low pH values ​​will also affect the ionization of organic compounds in the culture medium, thereby indirectly having an adverse effect on the growth and metabolism of microorganisms.

[0129] In summary, when the carbon source is sodium citrate, C / N is 30, the nitrogen source concentration is 100 mg / L, the salinity is 1%-12%, and the pH is 6.00, under these conditions, the composite bacterial agent NH4 + -N removal rate is the best and the growth of composite bacterial agent is the best, so the above factors can be selected as the conditions for microbial treatment of marine aquaculture tail water treatment tank.

[0130] The embodiments described above are only preferred specific implementations of the present invention. This specification uses the phrases "in one embodiment", "in another embodiment", "in yet another embodiment" or "in other embodiments" to refer to one or more of the same or different embodiments according to the present disclosure. Common changes and substitutions made by those skilled in the art within the scope of the technical solution of the present invention should be included in the protection scope of the present invention.

Claims

1. A salt-tolerant heterotrophic nitrifying composite bacterial agent, characterized in that: It is compounded by the seed liquid of alkaliphilic halomonas LJK2, Marinobacter LJK14 and alkaliphilic halomonas LJK7. The alkaliphilic halomonas LJK2 is deposited in the Guangdong Microbial Culture Collection Center on December 23, 2021, and the preservation number is GDMCC NO: 62157. The Marinobacter LJK14 is deposited in the Guangdong Microbial Culture Collection Center on December 23, 2021, and the preservation number is GDMCC No: 62156. The alkaliphilic halomonas LJK7 is deposited in the Guangdong Microbial Culture Collection Center on May 17, 2022, and the preservation number is GDMCC No: 62477; the volume ratio of the seed liquid of the alkaliphilic halomonas LJK2, the Marinobacterium LJK14 and the alkaliphilic halomonas LJK7 is 2.0-2.5:1.5-2.0:1.0-1.

5.

2. The salt-tolerant heterotrophic nitrifying composite bacterial agent according to claim 1, characterized in that: The volume ratio of the alkaliphilic halomonas LJK2, the Marinobacterium LJK14 and the alkaliphilic halomonas LJK7 seed liquid is 2.1-2.3:1.7-1.9:1.1-1.

3.

3. The salt-tolerant heterotrophic nitrifying composite bacterial agent according to claim 2, characterized in that: The volume ratio of the seed solutions of the alkaliphilic Halomonas LJK2, the Marinobacterium LJK14 and the alkaliphilic Halomonas LJK7 is 2.2:1.8:1.

2.

4. The salt-tolerant heterotrophic nitrifying composite bacterial agent according to any one of claims 1 to 3, characterized in that: The preparation method of the seed solution of each strain is as follows: the corresponding strain is inoculated into LB medium, cultured in a shaking table at 25-35°C and 100-200 rpm for 20-30 h, the bacterial suspension is centrifuged in a refrigerated centrifuge at 3000-5000 rpm for 5-15 min, the supernatant is removed, the sediment is washed with sterile water, and the supernatant is removed by centrifugation again, and the OD of the bacterial suspension is adjusted with sterile water. 600 The concentration was adjusted to 0.6-0.8 to obtain the seed solution of the strain.

5. Use of the salt-tolerant heterotrophic nitrifying composite bacterial agent as claimed in any one of claims 1 to 4 in reducing the ammonia nitrogen content in high-salt water bodies.

6. The use according to claim 5, characterized in that: The application comprises the following steps: adding a salt-tolerant heterotrophic nitrification composite bacterial agent and a carbon source into a high-salt water body with a pH of 6 to 8, wherein the volume ratio of the salt-tolerant heterotrophic nitrification composite bacterial agent to the water body is 3% to 15%, and culturing for 45h to 55h.

7. The use according to claim 6, characterized in that: Ammonia nitrogen concentration in high-salt water is 50-200 mg·L -1 .

8. The use according to claim 6, characterized in that: When carbon sources are added to high-salinity water bodies, the C / N ratio in the water body is 30-100.

9. The use according to claim 6, characterized in that: The carbon source is sodium citrate.

10. The use according to claim 6, characterized in that: The salinity of the high-salinity water body is 1% to 12%.

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